Ibragim Gaidarov

Spatial control of coated-pit dynamics in living cells

Here we visualize new aspects of the dynamics of endocytotic clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and clathrin light chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.

The Class II Phosphoinositide 3-Kinase C2α Is Activated by Clathrin and Regulates Clathrin-Mediated Membrane Trafficking

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.

Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.

Internalization of agonist‐activated G protein‐coupled receptors is mediated by non‐visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP‐2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestins function as an adaptor. High‐affinity binding sites for phosphoinositides in β‐arrestin (arrestin2) and arrestin3 (β‐arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported β2‐adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor–arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi‐point attachment of arrestin to the plasma membrane.

A Functional Phosphatidylinositol 3,4,5-Trisphosphate/Phosphoinositide Binding Domain in the Clathrin Adaptor AP-2 α Subunit. IMPLICATIONS FOR THE ENDOCYTIC PATHWAY

Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4,5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble D-myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 α subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 α sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian α, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4,5-trisphosphate to AP-2 inhibits the proteins clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.

Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.

Individual Phosphoinositide 3-Kinase C2α Domain Activities Independently Regulate Clathrin Function

Phosphoinositide 3-kinase C2α (PI3K-C2α) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2α was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2α, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2α in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2α can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2α employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.

Major histocompatibility complex class I-intercellular adhesion molecule-1 association on the surface of target cells: implications for antigen presentation to cytotoxic T lymphocytes

Polarization and segregation of the T‐cell receptor (TCR) and integrins upon productive cytotoxic T‐lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC‐I) and intercellular adhesion molecule‐1 (ICAM‐1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen‐A2 (HLA‐A2) MHC‐I and ICAM‐1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA‐A2 and ICAM‐1, induced the accumulation of membrane rafts, and beads loaded with ICAM‐1‐specific antibodies caused the selective recruitment of HLA‐A2 MHC‐I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA‐A2 and ICAM‐1 from the raft fraction, abatement of HLA‐A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca2+ flux in virus‐specific CTLs. These data suggest that productive engagement of ICAM‐1 on target cells facilitates the polarization of MHC‐I at the CTL–target cell interface, augmenting presentation of cognate peptide–MHC (pMHC) complexes to CTLs. We propose that ICAM‐1–MHC‐I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation.

Phosphoinositide 3-Kinase C2α Links Clathrin to Microtubule-dependent Movement

Phosphoinositide 3-kinase C2α (PI3K-C2α) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2α inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2α and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5–20 μm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2α accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2α structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2α and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2α and the microtubule motor machinery, with implications for membrane trafficking in intact cells.

Kinetics of 5-HT2B Receptor Signaling: Profound Agonist-Dependent Effects on Signaling Onset and Duration

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

Discovery of APD334: Design of a Clinical Stage Functional Antagonist of the Sphingosine-1-phosphate-1 Receptor.

APD334 was discovered as part of our internal effort to identify potent, centrally available, functional antagonists of the S1P1 receptor for use as next generation therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases. APD334 is a potent functional antagonist of S1P1 and has a favorable PK/PD profile, producing robust lymphocyte lowering at relatively low plasma concentrations in several preclinical species. This new agent was efficacious in a mouse experimental autoimmune encephalomyelitis (EAE) model of MS and a rat collagen induced arthritis (CIA) model and was found to have appreciable central exposure.